Although phosphorus (P) is an essential nutrient for biological productivity, it can cause freshwater degradation when present at fairly low concentrations. Monitoring studies using continuous sampling is crucial for documenting P dynamics in freshwater ecosystems and to reduce the risk of eutrophication. Despite literature updates of developments of the analytical methods for measurement of P species in natural waters, there has been no comprehensive review addressing freshwater sample collection, sample preparation, and sample treatment to fractionate and characterize different forms of P. Therefore, this paper aims to elaborate the different techniques for freshwater sampling and to introduce alternative laboratory methods for sample preservation and P fractionation. The advantages and disadvantages of various sampling techniques, including the traditional manual and the recently developed automatic and passive methods, are presented to highlight the importance of collecting representative freshwater samples. Furthermore, we provide suggestions for sample pretreatment, including filtration, transportation, and storage steps to minimize microbial activity and to maximize the accuracy of measurement of various P fractions. Finally, the most common laboratory methods to measure dissolved and particulate as well as the organic and inorganic freshwater P fractions are efficiently provided. Using this guide, a comprehensive monitoring program of P dynamics in freshwater ecosystems can be developed and applied to improve water quality, particularly of P-rich freshwaters.